Canine influenza virus and related compositions and methods of use

ABSTRACT

The present invention provides an isolated canine influenza virus of subtype H3N8 comprising an HA having SEQ ID NO: 4 or an amino acid sequence that is greater than 99% identical to SEQ ID NO: 4, with the proviso that the amino acids at positions 94 and 233 are identical to SEQ ID NO: 4; a composition comprising attenuated or inactivated virus; isolated or purified HA, NM, NP, M1, NS1, PA, PB1, and PB2 proteins and fragments thereof and compositions comprising same or nucleic acids, optionally as part of a vector, encoding same; and a method of inducing an immune response to canine influenza virus in an animal comprising administering to the animal an aforementioned composition.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional of U.S. Non-Provisional patentapplication Ser. No. 11/539,123, filed Oct. 5, 2006, now issued as U.SPat. No. 7,468,187, which claims the benefit of U.S. Provisional PatentApplication No. 60/727,808, filed Oct. 18, 2005, the contents of boththe applications are incorporated herein by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the fields of virology, molecularbiology, and immunology. In particular, the present invention relates tocanine influenza virus, as well as related compositions and methods ofuse in inducing an immune response in animals.

BACKGROUND OF THE INVENTION

Influenza virus is an RNA virus belonging to the familyOrthomyxoviridae. The viral RNA consists of eight independent segments,which easily recombine among influenza viruses to produce new subtypes.

Nucleoprotein (NP), which is the primary component of the nucleocapsid,is encoded in the fifth segment. The NP and the matrix protein are usedto classify the influenza virus into group A, B or C. Since NP is aninternal protein, it is not subject to the pressure of selection by ahost's immune system. It binds RNA, is part of the transcriptasecomplex, and is involved in the nuclear-cytoplasmic transport of viralRNA (vRNA).

Neuraminidase (NM), which splits the α-keto bond that joins a terminalsialic acid and the next sugar residue, thereby allowing the release ofviral progeny from infected cells, is encoded by the sixth segment. Ninesubtypes (N1-N9) of this enzyme have been identified. All subtypes havetwo structural regions—a stalk and a head. All N8 proteins have 470amino acids, the first eight of which are highly conserved. Thefollowing region is rich in hydrophobic amino acids and is considered tobe the transmembrane domain. The next 51 amino acids make up the stalkregion, and the head region begins at Cys91. The last region containsthe catalytic site of the enzyme. Cysteine residues in the head andstalk region tend to be highly conserved. There are 6-8 putativeN-glycosylation sites.

Hemagglutinin (HA), which is a membrane glycoprotein responsible for theadsorption of the virus into the host cell, is the main antigen to whichneutralizing antibodies are directed. Its antigenic variation is themajor cause of influenza epidemics. It is encoded by the fourth segment.Sixteen different subtypes (H1-H16) have been identified. HA has asignal peptide of 16 amino acids and two polypeptides (HA1 and HA2)joined by disulfide bridges. HA1 has the amino terminal end, whereas HA2has the carboxyl terminal end. A hydrophobic region in HA2 anchors HA tothe viral membrane. Cysteine residues tend to be highly conserved. Thereare six putative glycosylation sites, which enable the virus to mask itsantigenic sites (Skehel et al., PNAS USA 81: 1779 (1984)).

Other proteins include matrix (M or M1 and M2), non-structural (NS orNS1 and NS2), PA, PB1, and PB2. The M1 protein is a major component ofthe virion that binds to the plasma membrane of infected cells by meansof two hydrophobic regions at the N-terminus of the protein, whereas M2is an ion channel and, therefore, an integral membrane protein. The NS1protein is found in the nucleus and affects cellular RNA transport,splicing, and translation. The NS2 protein is found in the nucleus andcytoplasm and has unknown function. The PA protein is a transcriptaseand may have protease activity, whereas the PB1 protein functions intranscription elongation and the PB2 protein functions in transcriptioncap binding.

Globally, influenza is the most economically significant respiratorydisease in humans, pigs, horses and poultry (Wright et al.,Orthomyxoviruses. In: Fields Virology. Knipe et al., eds. LippincottWilliams & Wilkins, Philadelphia, 2001. pp. 1533-1579.). Influenza virusis known for its continuous genetic and antigenic changes, which impedeeffective control of the virus (Wright et al. (2001), supra; Webster etal., Microbiol. Rev. 56: 152-179 (1992)). Of particular concern forprevention of epidemics and pandemics is the emergency of a new subtypeof the virus by genetic re-assortment or inter-species transmission(Wright et al. (2001), supra).

Recently, influenza outbreaks have occurred in species, e.g., feline andcanine, which historically do not carry influenza virus (Keawcharoen etal., Emerg. Infect. Dis. 10: 2189-2191 (2004); Crawford et al., Science310: 398-485 (Oct. 21, 2005; published online Sep. 29, 2005); Dubovi etal., Isolation of equine influenza virus from racing greyhounds withfatal hemorrhagic pneumonia. In: Proceedings of the 47th Annual Meetingof American Association of Veterinary Laboratory Diagnosticians,Greensboro, N.C., Oct. 2005. p. 158; and Yoon et al., Emerg. Infect.Dis. 11(12): 1974-1976 (Dec. 2005)). Therefore, the host range ofinfluenza virus is expanding.

Outbreaks of respiratory disease in racing greyhounds caused byinfection with influenza virus have occurred in Florida in 2004, ineastern and western Iowa in April 2005, and in Texas in 2005. Thedisease was characterized by rapid onset of fever and cough, rapidrespiration, and hemorrhagic nasal discharge. The morbidity was almost100% in both race track compounds in Iowa, although the mortality wasless than 5%. While a large percentage of affected dogs recovered, manysuccumbed to hemorrhagic pneumonia. Therapeutic administration ofbroad-spectrum antibiotics reduced the severity of the disease but couldnot control it.

In view of the above, it is an object of the present invention toprovide the influenza virus that infects canines. It is another objectof the present invention to provide materials and methods for inducingan immune response to the influenza virus in canines. These and otherobjects and advantages, as well as additional inventive features, willbecome apparent from the detailed description provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an isolated canine influenza virus ofsubtype H3N8 comprising an HA having SEQ ID NO: 4 or an amino acidsequence that is greater than 99% identical to SEQ ID NO: 4, with theproviso that the amino acids at positions 94 and 233 are identical toSEQ ID NO: 4. In particular, the present invention provides an isolatedcanine influenza virus of subtype H3N8 deposited with the American TypeCulture Collection (Manassas, Va.) on Jun. 29, 2006, as Patent DepositNo. PTA-7694. Accordingly, the present invention also provides acomposition comprising attenuated virus as well as a compositioncomprising inactivated virus.

The present invention also provides isolated or purified proteins. Inone embodiment, the present invention provides an isolated or purifiedHA, which (i) has the amino acid sequence of SEQ ID NO: 4 or (ii) isderived from an influenza virus and which has an amino acid sequencethat is greater than 99% identical to SEQ ID NO: 4, with the provisothat the amino acid sequence is identical to that of SEQ ID NO: 4 atamino acid positions 94 and 233, or a fragment of (i) or (ii), whereinthe fragment comprises at least nine contiguous amino acids, at leastone of which is identical to the amino acid at position 94 or 233 of SEQID NO: 4.

In another embodiment, the present invention provides an isolated orpurified NM, which (i) comprises the amino acid sequence of SEQ ID NO: 2or (ii) is derived from an influenza virus and which comprises an aminoacid sequence that is greater than 99% identical to SEQ ID NO: 2, withthe proviso that the amino acid sequence is identical to that of SEQ IDNO: 2 at amino acid positions 68 and 134, or a fragment of (i) or (ii),wherein the fragment comprises at least nine contiguous amino acids, atleast one of which is identical to the amino acid at position 68 or 134of SEQ ID NO: 2.

In yet another embodiment, the present invention provides an isolated orpurified NP, which (i) has the amino acid sequence of SEQ ID NO: 6 or(ii) is derived from an influenza virus and which has an amino acidsequence that is greater than 99% identical to SEQ ID NO: 6, with theproviso that the amino acid sequence is identical to that of SEQ ID NO:6 at amino acid position 402, or a fragment of (i) or (ii), wherein thefragment comprises at least nine contiguous amino acids, at least one ofwhich is identical to the amino acid at position 402 of SEQ ID NO: 6.

In still yet another embodiment, the present invention provides anisolated or purified M1, which (i) has the amino acid sequence of SEQ IDNO: 8 or (ii) is derived from an influenza virus and which has an aminoacid sequence that is greater than 99% identical to SEQ ID NO: 8, withthe proviso that the amino acid sequence is identical to that of SEQ IDNO: 8 at amino acid position 111, or a fragment of (i) or (ii), whereinthe fragment comprises at least nine contiguous amino acids, at leastone of which is identical to the amino acid at position 111 of SEQ IDNO: 8.

Also provided is an isolated or purified NS1, which has the amino acidsequence of SEQ ID NO: 10.

Further provided is an isolated or purified PA protein, which (i) hasthe amino acid sequence of SEQ ID NO: 12 or (ii) is derived from aninfluenza virus and which has an amino acid sequence that is greaterthan 98% (or 99%) identical to SEQ ID NO: 12, with the proviso that theamino acid sequence is identical to that of SEQ ID NO: 12 at amino acidpositions 233, 256, 327, and 561, or a fragment of (i) or (ii), whereinthe fragment comprises at least nine contiguous amino acids, at leastone of which is identical to the amino acid at position 233, 256, 327,and 561, of SEQ ID NO: 12.

Still further provided is an isolated or purified PB1, which (i) has theamino acid sequence of SEQ ID NO: 14 or (ii) is derived from aninfluenza virus and which has an amino acid sequence that is greaterthan 99% identical to SEQ ID NO: 14, with the proviso that the aminoacid sequence is identical to that of SEQ ID NO: 14 at amino acidpositions 200 and 213, or a fragment of (i) or (ii), wherein thefragment comprises at least nine contiguous amino acids, at least one ofwhich is identical to the amino acid at position 200 or 213 of SEQ IDNO: 14.

Even still further provided is an isolated or purified PB2, which (i)has the amino acid sequence of SEQ ID NO: 16 or (ii) is derived from aninfluenza virus and which has an amino acid sequence that is greaterthan 99% identical to SEQ ID NO: 16, with the proviso that the aminoacid sequence is identical to that of SEQ ID NO: 16 at amino acidpositions 107, 221, 292, and 661, or a fragment of (i) or (ii), whereinthe fragment comprises at least nine contiguous amino acids, at leastone of which is identical to the amino acid at position 107, 221, 292,or 661 of SEQ ID NO: 16.

In view of the above, the present invention further provides acomposition comprising an above-described protein, such as HA or NM, ora fragment thereof in an amount sufficient to induce an immune responsein an animal and a biologically acceptable carrier.

Also in view of the above, the present invention provides a method ofinducing an immune response to canine influenza virus in an animal. Themethod comprises administering to the animal the composition comprisinga protein or fragment thereof.

An isolated or purified nucleic acid encoding above-described protein orfragment thereof, optionally as part of a vector, is also provided, asis a composition comprising the isolated or purified nucleic acid, whichexpresses the protein, such as HA or NM, or a fragment thereof, in anamount sufficient to induce an immune response in an animal and abiologically acceptable carrier.

Accordingly, the present invention also provides another method ofinducing an immune response to canine influenza virus in an animal. Themethod comprises administering to the animal the composition comprisinga nucleic acid.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is the partial nucleotide sequence (SEQ ID NO: 1; see alsoGenBank Acc. No. DQ146420) of the coding domain sequence (CDS) of the NMgene from subtype H3N8 of canine influenza virus. In accordance withconvention, the sequence is presented from left to right and top tobottom.

FIG. 2 is the amino acid sequence (SEQ ID NO: 2; see also GenBank Acc.No. DQ146420) encoded by SEQ ID NO: 1. In accordance with convention thesequence is presented in single letter format from left to right and topto bottom.

FIG. 3 is the complete nucleotide sequence (SEQ ID NO: 3; see alsoGenBank Acc. No. DQ146419) of the CDS of the HA gene from subtype H3N8of canine influenza virus.

FIG. 4 is the amino acid sequence (SEQ ID NO: 4; see also GenBank Acc.No. DQ146419) encoded by SEQ ID NO: 3.

FIG. 5 is the complete nucleotide sequence (SEQ ID NO: 5) of the CDS ofthe NP gene from subtype H3N8 of canine influenza virus.

FIG. 6 is the deduced amino acid sequence (SEQ ID NO: 6) encoded by SEQID NO: 5.

FIG. 7 is the complete nucleotide sequence (SEQ ID NO: 7) of the CDS ofthe M1 protein gene from subtype H3N8 of canine influenza virus.

FIG. 8 is the deduced amino acid sequence (SEQ ID NO: 8) encoded by SEQID NO: 7.

FIG. 9 is the complete nucleotide sequence (SEQ ID NO: 9) of the CDS ofthe NS1 protein gene from subtype H3N8 of canine influenza virus.

FIG. 10 is the deduced amino acid sequence (SEQ ID NO: 10) encoded bySEQ ID NO: 9.

FIG. 11 is the complete nucleotide sequence (SEQ ID NO: 11) of the CDSof the PA protein gene from subtype H3N8 of canine influenza virus.

FIG. 12 is the deduced amino acid sequence (SEQ ID NO: 12) encoded bySEQ ID NO: 11.

FIG. 13 is the complete nucleotide sequence (SEQ ID NO: 13) of the CDSof the PB1 protein gene from subtype H3N8 of canine influenza virus.

FIG. 14 is the deduced amino acid sequence (SEQ ID NO: 14) encoded bySEQ ID NO: 13.

FIG. 15 is the complete nucleotide sequence (SEQ ID NO: 15) of the CDSof the PB2 protein gene from subtype H3N8 of canine influenza virus.

FIG. 16 is the deduced amino acid sequence (SEQ ID NO: 16) encoded bySEQ ID NO: 15.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated on the discovery of a strain ofinfluenza virus in canines. The strain was isolated from racinggreyhounds in eastern and western Iowa. The strain has been classifiedas an H3N8 subtype, and has been designated A/canine/Iowa/13628/2005.Accordingly, the present invention provides a virus comprising an HAhaving SEQ ID NO: 4 or an amino acid sequence that is greater than 99%identical to SEQ ID NO: 4, with the proviso that the amino acids atpositions 94 and 233 are identical to SEQ ID NO: 4. The virus canfurther comprise an NM comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence that is greater than 99% identical to SEQ IDNO: 2, with the proviso that the amino acids at positions 68 and 134 areidentical to SEQ ID NO: 2. The virus comprising the aforementioned HA,alone or in further combination with the aforementioned NM, can furthercomprise at least one of the following: an NP having the amino acidsequence of SEQ ID NO: 6 or an amino acid sequence that is greater than99% identical to SEQ ID NO: 6, with the proviso that amino acid 402 isidentical to that of SEQ ID NO: 6; an M1 having the amino acid sequenceof SEQ ID NO: 8 or an amino acid sequence that is greater than 99%identical to SEQ ID NO: 8, with the proviso that amino acid 111 isidentical to that of SEQ ID NO: 8; an NS1 having the amino acid sequenceof SEQ ID NO: 10; a PA protein having the amino acid sequence of SEQ IDNO: 12 or an amino acid sequence that is greater than 98% (or 99%)identical to SEQ ID NO: 12, with the proviso that amino acids 233, 256,327, and 561 are identical to SEQ ID NO: 12; a PB1 having the amino acidsequence of SEQ ID NO: 14 or an amino acid sequence that is greater than99% identical to SEQ ID NO: 14, with the proviso that amino acids 200and 213 are identical to SEQ ID NO: 14; and/or PB2 having the amino acidsequence of SEQ ID NO: 16 or an amino acid sequence that is greater than99% identical to SEQ ID NO: 16, with the proviso that amino acids 107,221, 292, and 661 are identical to SEQ ID NO: 16. In particular, thepresent invention provides an isolated canine influenza virus of subtypeH3N8 deposited with the American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209, U.S.A., on Jun. 29, 2006, asPatent Deposit No. PTA-7694.

Influenza virus can be precipitated by subjecting the virus in aqueousmedium to one or more insolubilizing steps brought about by the presenceof up to 5% by weight of polyethylene glycol (PEG) having a molecularweight between 3,000 and 20,000 or another linear filamentarynon-charged polymer in an amount equivalent to the solubilizing power ofPEG, separating an insolublized fraction from a non-insolubilizedfraction, and recovering virus from one of the fractions (see, e.g.,U.S. Pat. No. 3,989,818). Preferably, the temperature does not exceed35° C., the pH is between 6 and 9, and the ionic strength of the aqueousmedium is below the salting out point for the virus. The concentrationof the virus in the aqueous medium prior to insolubilizing correspondsto a hemagglutination titer of at least 1 in 32. Aggregated viralparticles are obtained, which are believed to provide a better antigeniceffect due to the slow release of viral particles after vaccination. If,however, non-aggregated or less aggregated particles are desired, theycan be dissociated using any suitable method, such as sonication.

The virus can be attenuated by passaging in a cell system until thevirus has lost its ability to produce disease, while fully retaining itsimmunogenic character. For example, the virus can be serially passagedin a culture of cells originating from a canine species or othersuitable species at a temperature of about 37° C. At each passage, thevirus is harvested from one culture and inoculated into a mediumcontaining a fresh cell culture in accordance with methods known in theart. For example, the virus can be collected from tissue cell culturefluids and/or cells. Optionally, during harvesting, the cell culture canbe sonicated to promote release of the virus. See, e.g., U.S. Pat. Nos.5,698,433 and 6,455,298.

If desired, an influenza strain can be passaged at least once in theallantoic cavity of embryonated eggs, such as chicken eggs, in thepresence of serum, to obtain serum-resistant virus (see, e.g., U.S. Pat.No. 3,953,592; Kilbourne et al., J. Exp. Med. 111: 387 (1960);Kilbourne, Science 160: 74-75 (April 1968); and Layer et al., Virology30: 493-501 (1966)). High potency influenza vaccine with lowpyrogenicity and low endotoxicity can be achieved by treating theconcentrated allantoic fluid containing an attenuated virus sequentiallywith butyl acetate and ethyl acetate, followed by flash evaporation(see, e.g., U.S. Pat. No. 4,000,257). Such virus can be administeredintranasally as a vaccine.

Once inoculated into the host, the virus multiplies to some extent sothat only a small initial inoculum is required. The virus must beinnocuous, and infection of susceptible contacts should be kept to aminimum.

Alternatively, the virus can be inactivated by abolishing replicationand virulence. This can be done by chemical or physical means. Chemicalinactivation can be carried out by treatment of the virus with anenzyme, formaldehyde, β-propiolacton or derivative thereof,ethyleneimine or derivative thereof, an organic solvent (e.g.,halogenated hydrocarbon), and/or a detergent (e.g., Tween®, Triton X®,sodium desoxycholate, sulfobetain, or cetyltrimethylammonium salts). Ifnecessary, chemically activated compositions can be neutralized. Forexample, if formaldehyde is used to deactivate the composition, thecomposition can be neutralized with thiosulphate. If required, the pHsubsequently can be returned to a value of about 7. Alternatively, thevirus can be extracted with a mixture of ether and ethanol, the aqueousand organic phases can be separated, and residual ether can be removedfrom the viral suspension under reduced pressure (see, e.g., U.S. Pat.No. 4,431,633). Physical inactivation advantageously can be carried bysubjecting the virus to energy-rich radiation, such as ultravioletlight, γ-radiation, or X-rays. Inactivated forms require a relativelyhigh amount of inoculum and, therefore, a correspondingly large quantityof antigenic material, which has to be manufactured, tested, anddistributed.

In view of the above, the present invention also provides a compositioncomprising an attenuated or inactivated virus. The virus should bepresent in an amount sufficient to induce an immune response and,desirably, should provide protection upon challenge. Generally, anadjuvant, such as Tween®, Span®, Freund's complete adjuvant, saponin,Corynebacterium parvum (Coparvax®), aluminium phosphate, aluminiumhydroxide, or a mixture thereof, is added to the composition,particularly if the composition comprises inactivated virus. Proteinhydrolysates and/or amino acids can be added to stabilize thecomposition (see, e.g., U.S. Pat. No. 4,537,769). Alternatively, thecomposition can be formulated as an oil-in-water emulsion using oilssuch as Marcol and/or Arlacel.

Recombinant influenza strains also can be prepared, such as from thecombination of an “over-attenuated” (i.e., the number of passages forattenuation is substantially greater than what is normally required toremove pathogenicity) influenza A parent strain, e.g., A2, with avirulent influenza strain as provided herein (see, e.g., U.S. Pat. No.3,991,179; also, see U.S. Pat. Nos. 4,009,258; 4,278,662; 4,318,903;4,338,296; and 4,693,893). A recombinant strain preferably has thegrowth characteristics of the over-attenuated strain coupled with theantigenic properties, e.g., the HA and NM proteins, of the virulentstrain. The selection of strains of influenza virus for vaccineformulation is described in U.S. Pat. No. 5,162,112. Recombinant strainscan be formulated as compositions for inducing an immune response.

Sucrose, arginine monohydrochloride, the monosodium monohydrate ofglutamic acid, and gelatin hydrolysate can be used to stabilize aninfluenza virus composition for storage in a refrigerator. See, e.g.,U.S. Pat. App. Pub. No. 2006/0110406.

In view of the above, the present invention also provides an isolated orpurified HA. The HA either has the amino acid sequence of SEQ ID NO: 4or is derived from an influenza virus and has an amino acid sequencethat is greater than 99% identical to SEQ ID NO: 4, with the provisothat the amino acid sequence is identical to that of SEQ ID NO: 4 atamino acid positions 94 and 233. A fragment of HA comprising at leastnine (such as 9, 12, 15, 18, 21 or 24) contiguous amino acids, at leastone of which is identical to the amino acid at position 94 or 233 of SEQID NO: 4, is also provided.

An isolated or purified NM is also provided. The NM comprises the aminoacid sequence of SEQ ID NO: 2 or is derived from an influenza virus andcomprises an amino acid sequence that is greater than 99% identical toSEQ ID NO: 2, with the proviso that the amino acid sequence is identicalto that of SEQ ID NO: 2 at amino acid positions 68 and 134. A fragmentof NM comprising at least nine contiguous amino acids, at least one ofwhich is identical to the amino acid at position 68 or 134 of SEQ ID NO:2, is also provided.

Further provided is an isolated or purified NP. The NP has the aminoacid sequence of SEQ ID NO: 6 or is derived from an influenza virus andhas an amino acid sequence that is greater than 99% identical to SEQ IDNO: 6, with the proviso that the amino acid sequence is identical tothat of SEQ ID NO: 6 at amino acid position 402. A fragment of NPcomprising at least nine contiguous amino acids, at least one of whichis identical to the amino acid at position 402 of SEQ ID NO: 6, is alsoprovided.

Still further provided is an isolated or purified M1. The M1 has theamino acid sequence of SEQ ID NO: 8 or is derived from an influenzavirus and has an amino acid sequence that is greater than 99% identicalto SEQ ID NO: 8, with the proviso that the amino acid sequence isidentical to that of SEQ ID NO: 8 at amino acid position 111. A fragmentof M1 comprising at least nine contiguous amino acids, at least one ofwhich is identical to the amino acid at position 111 of SEQ ID NO: 8, isalso provided.

Even still further provided is an isolated or purified NS1, which hasthe amino acid sequence of SEQ ID NO: 10.

An isolated or purified PA protein is also provided. The PA has theamino acid sequence of SEQ ID NO: 12 or is derived from an influenzavirus and has an amino acid sequence that is greater than 98% (or 99%)identical to SEQ ID NO: 12, with the proviso that the amino acidsequence is identical to that of SEQ ID NO: 12 at amino acid positions233, 256, 327, and 561. A fragment of PA comprising at least ninecontiguous amino acids, at least one of which is identical to the aminoacid at position 233, 256, 327, or 561 of SEQ ID NO: 12, is alsoprovided.

An isolated or purified PB 1 is provided. The PB1 has the amino acidsequence of SEQ ID NO: 14 or is derived from an influenza virus and hasan amino acid sequence that is greater than 99% identical to SEQ ID NO:14, with the proviso that the amino acid sequence is identical to thatof SEQ ID NO: 14 at amino acid positions 200 and 213. A fragment of PB1comprising at least nine contiguous amino acids, at least one of whichis identical to the amino acid at position 200 or 213 of SEQ ID NO: 14,is also provided.

Provided also is an isolated or purified PB2. The PB2 has the amino acidsequence of SEQ ID NO: 16 or is derived from an influenza virus and hasan amino acid sequence that is greater than 99% identical to SEQ ID NO:16, with the proviso that the amino acid sequence is identical to thatof SEQ ID NO: 16 at amino acid positions 107, 221, 292, and 661. Afragment of PB2 comprising at least nine contiguous amino acids, atleast one of which is identical to the amino acid at position 107, 221,292, or 661 of SEQ ID NO: 16, is provided as well.

The above proteins and fragments thereof can be purified (coupled withchemical or physical fragmentation to generate fragments) or synthesizedin accordance with methods known in the art. See, e.g., Meienhofer,Hormonal Proteins and Peptides 2: 46, Academic Press, NY (1973), forsolid phase protein synthesis, and Schroder et al., The Peptides, vol.1, Academic Press, NY (1965), for solution phase protein synthesis.Automated systems can be used to carry out such techniques in accordancewith manufacturer's instructions. Therapeutic quantities can berecombinantly produced and purified.

Alternatively, proteins, in particular HA and NM, can be isolated byselective solubilization, while leaving residual subviral particlesconsisting of the intact lipid/protein membrane enclosing all othernon-essential viral components. The difference in size/density of thesolubilized proteins and the residual subviral particles allowsseparation based on differences in physical properties by gradientcentrifugation and fractionation, sedimentation, molecular sievechromatography, or pelleting in an ultracentrifuge. Selectivesolubilization of HA and NM can be achieved by treatment of the viruswith a cationic detergent (see, e.g., U.S. Pat. No. 4,140,762; the '762patent). The whole virus-containing fluid obtained from cell culture canbe treated with a DNA-digesting enzyme followed by addition of acationic detergent and isolation of surface-antigen proteins (see, e.g.,U.S. Pat. No. 5,948,410). The fluid can be subjected to severalultracentrifugation steps, or the virus can be fragmented in thepresence of an amphiphilic nonionic detergent followed by filtration toremove undesirable substances (see, e.g., U.S. Pat. No. 6,048,537).Alternatively, membrane filtration and chemical splitting can be used toobtain a viral protein (see, e.g., U.S. Pat. No. 4,327,182). Otherprocedures are described in U.S. Pat. Nos. 4,064,232 and 4,057,626.Preferably, the virus is multiplied before treatment as exemplified inthe '762 patent (col. 2, 11. 10 et seq).

Mapping can be conducted to identify an immune response-inducing epitopeof a viral protein, i.e., “epitope mapping.” Such mapping involvesfragmenting of a protein into overlapping peptides (such as peptidescomprising 9, 12, 15, 18, 21 or 24 amino acids). The protein can befragmented with a proteolytic enzyme. The individual peptides are thentested for their ability to bind to an antibody elicited by the nativeprotein or to induce T-cell or B-cell activation. Alternatively,hydrophilic regions of the protein can be selected, since hydrophilicresidues are often on the surface of the protein and, therefore, areaccessible to the antibody. X-ray crystallographic analysis of theantigen-antibody complex also can be performed. Potential HLA anchorbinding motifs, which are peptide sequences that are known to be likelyto bind to MHC molecules, can be identified from the amino acid sequenceof a protein. Preferably, the epitope selected is one that shares littleto no sequence identity with sequences widely found in the animal towhich a composition comprising or expressing a protein fragment will beadministered.

An isolated or purified nucleic acid encoding an above-described proteinor fragment thereof, optionally as part of a vector, is also provided.The nucleic acid encoding the HA can comprise the nucleotide sequence ofSEQ ID NO: 3 or a fragment thereof encoding at least nine (9, 12, 15,18, 21 or 24) contiguous amino acids. If desired, a trivalent vaccinebased on HA can be prepared, wherein one of the HAs comprises the aminoacid sequence of SEQ ID NO: 4 (see, e.g., U.S. Pat. Nos. 5,762,939 and6,245,532; see, e.g., U.S. Pat. No. 6,740,325 for a tetravalentvaccine). The nucleic acid encoding the NM can have the nucleotidesequence of SEQ ID NO: 1 or a fragment thereof encoding at least ninecontiguous amino acids (see, e.g., U.S. Pat. No. 6,605,457 and U.S. Pat.App. Pub. No. 2003/0129197), whereas the nucleic acid encoding the NPcan have the nucleotide sequence of SEQ ID NO: 5 or a fragment thereofencoding at least nine contiguous amino acids, the nucleic acid encodingthe M1 protein can have the nucleotide sequence of SEQ ID NO: 7 or afragment thereof encoding at least nine contiguous amino acids, thenucleic acid encoding the NS1 protein can have the nucleotide sequenceof SEQ ID NO: 9, the nucleic acid encoding the PA can have thenucleotide sequence of SEQ ID NO: 11 or a fragment thereof encoding atleast nine contiguous amino acids, the nucleic acid encoding the PB1 canhave the nucleotide sequence of SEQ ID NO: 13 or a fragment thereofencoding at least nine contiguous amino acids, and the nucleic acidencoding the PB2 can have the nucleotide sequence of SEQ ID NO: 15 or afragment thereof encoding at least nine contiguous amino acids. One ofordinary skill in the art will appreciate, however, that due to thedegeneracy of the genetic code, there are numerous other nucleotidesequences that can encode such amino acid sequences.

The above nucleic acids, which can be DNA or RNA, and fragments thereofcan be synthesized (see, e.g., Oligonucleotide Synthesis, Gait, ed.,1984). Such molecules can include non-naturally occurringnucleotides/bases that encode the desired amino acid sequence. Forexample, the base or sugar can be methylated. In addition, the backboneof the nucleic acid molecule can be modified, e.g., a phosphorothioatebackbone, methylphosphonate, methylphosphorothioate, phosphorodithioate,and combinations thereof.

Alternatively, isolated vRNA can be subjected to reverse transcriptaseto produce an RNA/DNA hybrid, from which the RNA is digested away andthe residual DNA is treated to produce a dsDNA having a hairpin end,which is treated with a single-strand-specific nuclease to produce abimolecular double-stranded copy of the vRNA (see, e.g., U.S. Pat. No.4,357,421). See, e.g., U.S. Pat. App. Pub. No. 2006/0166321 for the useof tandem transcription cassettes for the preparation of influenza inthe absence of helper virus.

The nucleic acid is optionally part of a DNA vector comprising at leastone promoter, in which case each nucleotide sequence is operably linkedto a promoter, which can be the same or different. In addition topromoters, other control sequences, such as terminating signals and thelike, can be part of the DNA vector.

For example, the nucleic acid can be introduced into a suitablerecombinant expression vector, such as those adapted for bacteria, suchas E. coli and Salmonella typhi, yeast, such as Saccharomyces cervisiaeor Pichia pastoris, or filamentous fungi, such as Aspergillus nidulans.The bacteria, yeast, or fungi can be grown in continuous culture. Thepolypeptide, which is produced during culture, then can be isolated andpurified. Alternatively, the nucleic acid molecule can be introducedinto Poxyiridae (e.g., fowlpox-based vectors), Herpes-viridae (e.g.,pseudorabies virus-based vectors, turkey herpes virus-based vectors,feline herpes virus-based vectors, infectious laryngotracheitisvirus-based vectors, and bovine herpes virus-based vectors),Adenoviridae (e.g., bovine adenovirus (e.g., serotype 3), humanadenovirus (e.g., serotype 4 or 7), and canine adenovirus (e.g.,serotype 2; CAV2; see, e.g., U.S. Pat. No. 6,090,393), or an insectvirus expression vector, such as recombinant baculovirus (e.g.,Autographa californica nuclear polyhydrosis virus (AcNPV)), which, inturn, can be used to infect susceptible cultured SF9 cells, which arederived from the insect Spodotera frugiperda. Other viral vectorsinclude vaccinia (see, e.g., U.S. Pat. No. 4,722,848), adenovirus,adeno-like virus, adeno-associated virus, retro-virus, and pox (see,e.g., Hruby, Vet. Parasitol. 29: 281-282 (1988); Uiu, “AIDS ResearchReviews,” Dekker, Inc., 1991, 1: 403-416), which can be administered bya skin scratch or by injection, optionally as a liposomal formulation.Other vectors include Bacille-Calmette-Guerin (BCG; Stover et al.,Nature 351: 456-460 (1991)), detoxified anthrax toxin vectors, and thelike. Mammalian cells, such as Chinese hamster ovary (CHO) cells, andeven plant cells can be used to express the polypeptide from theappropriate construct. One of ordinary skill in the art will appreciatethat the choice of host cell will affect the nature ofpost-translational processing (e.g., glycosylation, folding, and thelike), which, in turn, can impact the immunogenicity of the polypeptide,and subsequent purification techniques.

Expression can be achieved in any appropriate host celltransformed/transfected with the expression vector. Examples of suitablehost cells include, but are not limited to, those described above. Thus,the present invention also provides a host cell transformed/transfectedwith an expression vector.

Supernatants from host/vector systems that secrete the protein orfragment thereof into culture media can be applied to a purificationmatrix, such as an affinity column or an ion exchange column. One ormore reverse-phase HPLC steps can be employed to purify further therecombinant protein or fragment thereof.

Production of a protein or fragment thereof as a fusion protein canstabilize production. This can be accomplished by ligatingpolynucleotide sequences encoding two or more proteins (or fragmentsthereof) into an appropriate expression vector with or without apeptidic linker. Desirably, the reading frames of the polynucleotidessequences are in phase, so that a single fusion protein that retains thebiological activity of each protein (or fragment thereof) is produced. Apeptidic linker from 1 to about 50 amino acids can be used to separatethe resultant proteins (or fragments thereof) so as to ensure that eachprotein (or fragment thereof) properly folds into its native secondary,tertiary, and quaternary structures (see, e.g., Maratea et al., Gene 49:39-46 (1985); Murphy et al., PNAS USA 83: 8258-8262 (1986); U.S. Pat.No. 4,935,233; and U.S. Pat. No. 4,751,180). The ability to adopt aflexible extended conformation, the inability to adopt a secondarystructure that could interact with functional amino acids on either oneor both of the proteins, and the lack of hydrophobic or charged residuesthat might react with either one or both of the proteins are factors,which are taken into consideration in selecting a peptide linker.Linkers are not required when the ends of the proteins to be joined donot contain essential regions, such that the ends can be used toseparate functional domains and prevent steric interference. Preferredpeptide linker sequences contain Gly, Asn, and Ser residues. Other nearneutral residues, such as Thr and Ala, also can be used.

Other additional amino acid sequence(s) can be selected to enhance theexpression and/or immunogenicity of the protein or fragment thereof. Forexample, the protein or fragment thereof can be fused to the heavy chainof immunoglobulin G (IgG) or an antigen-presenting cell (APC) bindingprotein or a dendritic cell binding protein, such as IL-D, GM-CSF, IL-1,TNF, IL-4, CD40L, CTLA4, CD28, or FLT-3 ligand. Techniques, such as theuse of dehydrating agents, e.g., dicyclohexylcarbodiimide (DCCI), or thecreation of linkages between sulfhydryl groups, epsilon amino groups,carboxyl groups, and the like, can be used. If desired, a cleavage sitecan be introduced into the fusion protein to enable separation of theprotein (or fragment thereof) from the non-naturally occurringsequence(s). Examples of cleavage sites include a target sequence for aproteolytic enzyme or, if methionine is not present in the protein (orfragment thereof), methionine, which, in turn, is cleaved by cyanogenbromide. Such methods are known in the art. The protein or fragmentthereof can be modified by glycosylation or other derivatization (e.g.,acetylation or carboxylation), also in accordance with methods known inthe art.

The protein (or fragment thereof) can be expressed in situ from asuitable expression system. Any DNA construct, which is effective inproducing the encoded protein or fragment thereof in the desiredenvironment, can be used to express the protein or fragment thereof asdescribed above.

Alternatively, the nucleic acid molecule can behave as an effectiveexpression system in situ when injected into an animal as “naked DNA”(see, e.g., Ulmer et al., Science 259: 1745-1749 (1993); and Cohen,Science 259: 1691-1692 (1993)). DNA delivery also can be facilitatedthrough the use of bupivicaine, polymers, and peptides; alternatively,cationic lipid complexes, particles, or pressure (see, e.g., U.S. Pat.No. 5,922,687) can be used.

Examples of amino acid sequences that are at least about or greater than95% identical to, such as at least about or greater than 96%, 97%, 98%,or 99% identical to, SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16 includeamino acid sequences that contain one or more substitutions, insertions,additions and/or deletions. Sequence identity can be determined byaligning polypeptide sequences and applying publicly available computeralgorithms, such as BLASTP (Pearson et al., PNAS USA 85: 2444-2448(1988); Pearson, Methods Enzymol. 183: 63-98 (1990); and Altschul etal., Nucl. Acids Res. 25: 3389-3402 (1997)). The software for BLASTP isavailable on the FTP server of the National Center for BiotechnologyInformation (NCBI) or NCBI, National Library of Medicine, Building 38A,Room 8N8O5, Bethesda, Md. 20894. Once the polypeptide sequences arealigned, the number of identical amino acids over the aligned portionsis identified, the number of identical amino acids is divided by thetotal number of amino acids of the polypeptide of interest, and theresult is multiplied by 100 to determine the percentage sequenceidentity.

In this regard, one of ordinary skill in the art will appreciate that afragment of a given amino acid sequence can be at least about or greaterthan 95% identical to, such as 96%, 97%, 98% or 99% identical to, theamino acid sequence. Thus, fragments are intended to be encompassed by“an amino acid sequence that is at least about or greater than 95% (or96%, 97%, 98% or 99%) identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or16. ” Such fragments desirably retain the immunogenicity of thefull-length protein. Functional fragments can be generated by mutationalanalysis of the nucleic acid encoding the protein and subsequentexpression of the resulting mutant protein or by chemical/enzymaticdigestion of the protein, itself.

Modifications, such as substitutions, insertions, additions and/ordeletions, can be introduced into the nucleic acid or the protein (orfragment thereof) in accordance with methods known in the art (see,e.g., Adelman et al., DNA 2: 183 (1983), for oligonucleotide-directedsite-specific mutagenesis). Desirably, the modification does notsubstantially diminish the immunogenicity of the protein fragment;rather, it is preferred that the immunogenicity remains substantiallythe same or increases relative to the unmodified protein.

A “conservative substitution” is one in which an amino acid issubstituted for another amino acid that has similar properties, i.e.,similar secondary structure and hydropathic nature. Amino acidsubstitutions can be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity and/or theamphipathic nature of the residues. For example, negatively chargedamino acids, such as aspartic acid and glutamic acid, can beinterchanged, whereas positively charged amino acids, such as lysine andarginine, can be interchanged, and amino acids with uncharged polar headgroups having similar hydrophilicity values can be interchanged. In thisregard, leucine, isoleucine and valine can be interchanged, glycine andalanine can be interchanged, asparagine and glutamine can beinterchanged, and serine, threonine, phenylalanine, and tyrosine can beinterchanged. Other groups of amino acids that can be interchangedinclude: (1) ala, pro, gly, glu, asp, gln, asn, ser and thr; (2) cys,ser, tyr and thr; (3) val, ile, leu, met, ala and phe; (4) lys, arg andhis; and (5) phe, tyr, trp, and his.

In view of the above, a composition comprising the isolated or purifiedprotein/nucleic acid or fragment of either of the foregoing and abiologically acceptable carrier is also provided. The nucleic acid orfragment thereof can be part of a vector. See, for example, U.S. Pat.No. 4,029,763, which is directed to an influenza vaccine comprising, asan active ingredient, NM, and U.S. Pat. No. 4,140,762, which is directedto an influenza vaccine comprising, as active ingredients, HA and NM.U.S. Pat. No. 4,826,687 describes the addition of muramyl dipeptide to avaccine comprising HA and NM. If desired, polypeptides correspondingsubstantially to amino acids 148-162, 163-166, and/or 215-239 of M1 canbe added to a composition of a protein/nucleic acid or fragment thereof(see, e.g., U.S. Pat. Nos. 5,136,019; 5,616,327; and 5,741,493). Anysuitable biologically acceptable carrier can be used in the composition.For example, the protein(s)/nucleic acid(s)/fragments thereof can beresuspended in a diluent, e.g., 0.9% sodium chloride solution, which isoptionally buffered with, for example, a phosphate buffer. Any sucrosethat remains from purification of the virus can be reduced by dialysis.Dialysis or gel chromatography can be used to remove any remainingcationic detergent. Preferably, the protein or fragment thereof ispresent in an amount sufficient to induce an immune response (i.e.,cellular or humoral) in an animal. A frequently selected carrier forpharmaceuticals and antigens is poly(d,l-lactide-co-glycolide) (PLGA).PLGA is a biodegradable polyester, and can be used for the controlledrelease of antigen (Eldridge et al., Curr. Topics Micro. Immuno. 146:59-66 (1989); see also U.S. Pat. No. 6,090,393). The entrapment ofantigens in PLGA microspheres of 1-10 μ in diameter has been shown tohave a remarkable adjuvant effect when administered orally.

If desired, a preserving agent or an inactivating agent, such asformaldehyde, can be added. A conventional amount ofpreserving/inactivating agent is 1 part per 10,000 parts.

If desired, one or more proteins (or immunogenic fragments thereof),such as the above-described HA, can be combined with proteosomes. See,e.g., U.S. Pat. No. 6,743,900 and U.S. Pat. App. Pub. No. 2004/0156867.

Immunogenicity can be improved by inclusion of conventionalimmunological adjuvants, such as aluminium hydroxide (e.g., about 0.2%)or aluminium phosphate, aluminum (see, e.g., U.S. Pat. Nos. 6,372,223,6,635,246, 6,861,244 and 7,052,701 and U.S. Pat. App. Pub. Nos.2004/0096464 and 2006/0147468), chitosan (see, e.g., U.S. Pat. Nos.6,136,606 and 6,534,065), alum, such as in the form of aluminumhydroxide, aluminum phosphate or aluminum oxide, mineral oils (e.g.,Bayol F® and Marcol 52®), Freund's complete adjuvant, Freund'sincomplete adjuvant, muramyl dipeptide, monophosphoryl lipid A, andsaponins, including the Quil A component. Immunogenicity also can beimproved by adding a cytokine, such as an interleukin, or by conjugatingproteins or fragments thereof. Preferably, the protein or fragmentthereof is conjugated with a macromolecular carrier, such as a protein(e.g., serum albumin, keyhole limpet hemocyanin, immunoglobulin,throglobulin, and ovalbumin), polysaccharide (e.g., latex-functionalizedsepharose, agarose, cellulose beads, and the like), phospholipid,polymeric amino acids (e.g., polyglutamic acid, polylysine, and thelike), or amino acid co-polymers (see, e.g., U.S. Pat. Nos. 5,136,019and 5,612,037). Alternatively, the protein or fragment thereof can beencapsulated with a proteoliposome or lipid vesicle.

The composition, which can induce an immune response, can be prepared inthe form of a suspension or can be lyophilized. If lyophilized, it ispreferable to add one or more stabilizers. Suitable stabilizers are, forexample, sucrose, phosphate, glutamate, and albumin (SPGA; Bovarnick, J.Bacteriol. 59: 509 (1950)), carbohydrates (e.g., sorbitol, mannitol,starch, dextran, and glucose), proteins (e.g., albumin and casein) ordegradation products thereof, protein-containing agents (e.g., bovineserum or skim milk), and buffers (e.g., alkali metal phosphates).

Alternatively, the composition can be formulated as a controlled-releasecomposition. The attenuated/inactivated virus or recombinant vector canbe microencapsulated with polymers, such as polycarbonates, polyesters,polyurethanes, polyorthoesters, and polyamides. The particular polymerselected depends on a number of factors including reproducibility ofpolymer synthesis and microencapsulation, cost of materials and process,toxicological profile, requirements for variable release kinetics, andthe physicochemical compatibility of the polymer and the virus/vector.

The compositions described herein can be used alone or in combinationwith other active ingredients/compositions. Examples includecompositions, which can induce an immune response again caninedistemper, infectious canine hepatitis (CAV-1 and CAV-2), rabies,parainfluenza, canine corona virus, measles, leptospirosis, andBordetella. Polyphenols have been disclosed to inhibit influenzainfection in humans (see, e.g., U.S. Pat. No. 5,173,922; the '922patent). Accordingly, the addition of a polyphenol, such asepigallocatechin gallate, epicatechin gallate, epigallocatechin,epicatechin, free theaflavin, theaflavin monogallate A, theaflavinmonogallate B, and/or theaflavin digallate may be beneficial (see the'922 patent). Inhibitors of NM are disclosed in U.S. Pat. No. 5,453,533.The use of cytokines as immunopotentiators and liposomal encapsulationare described in U.S. Pat. No. 5,919,480.

The amount of nucleic acid in the composition can vary widely. Forexample, the concentration can range from less than about 0.1% to asmuch as about 20-50% or more by weight, usually at least about 2%. Theconcentration of protein in the composition also can vary widely. Forexample, the concentration can range from less than about 0.1% to asmuch as about 20-50% or more by weight, usually at least about 2%. Fluidvolume and viscosity are taken into consideration when determining thefinal concentration.

Accordingly, a method of inducing an immune response to canine influenzavirus in an animal is also provided. The susceptibility of an animal toinfection can be assessed using the plaque reduction neutralization test(U.S. Pat. No. 4,315,073) or the hemagglutination test. The methodcomprises administering to the animal an above-described compositioncomprising an isolated or purified protein/nucleic acid or fragmentthereof. If the composition comprises a nucleic acid (or fragmentthereof) as part of a vector, preferably the protein (or fragmentthereof) is expressed in an amount sufficient to induce an immuneresponse in an animal. For example, a single dose of from about 9 toabout 43 international units per kg of animal body weight can beadministered. For larger mammals, a single dose can comprise from about600 to about 3,000 international units per kg of body weight. Forvaccine compositions prepared by culturing virus in the allantoic cavityof fertile eggs, harvesting the virus, and, if desired, stabilizing theharvested virus with a stabilizer, such as a peptone or sucrose, andthen distribution into glass vials for subsequent freeze-drying, aneffective vaccine dosage unit can contain at least 10⁷ EID50 (50%egg-infective dose) of virus. In the latter situation, the freeze-driedvaccine is reconstituted by addition of water or anotherpharmaceutically acceptable diluent prior to administration, such as inthe form of a nasal spray or nasal drops. If desired, the vaccine can beadministered in two successive dosages at a one-week interval.

The composition can be administered to puppies as a single dose at theage of 12 weeks, or repeatedly starting from the age of 6 weeks (e.g.,at 6, 9 and 12 weeks), or weekly from 4 weeks on. The effective dosageand route of administration are determined by the nature of thecomposition, the nature of the expression product, LD₅₀, and, ifrecombinant vector is used, the expression level of the vector, as wellas the breed of dog and its age, sex, weight, and condition. Dosages ofexpressed product can range from a few to a few hundred micrograms,e.g., 5-500 μg. Preferred dosages of virus or recombinant vector canrange from about 10³ to about 10⁶ pfu. The dose for the live attenuatedstrain can be at least about 10³ TCID₅₀.

The compositions can be administered parenterally (i.e., by injection(e.g., intradermal, subcutaneous, or intramuscular) or by the route ofinfection, such as nasally) or enterally (i.e., by oral administration).The use of a gelling agent and a muco- or bio-adhesive to enhance theimmune response against an intradermally administered immunogeniccomposition is described in U.S. Pat. App. Pub. No. 2005/0255121. Ifdesired, the composition for inducing an immune response can beadministered through drinking water or syrup in accordance with Chu etal. (U.S. Pat. App. Pub. No. 2006/0171960, which was published on Aug.3, 2006). Oral administration is advantageous inasmuch as it avoidstime-consuming and labor-intensive intramuscular injection, which, inturn, can create stress for the animal and discomfort. Discomfort, inturn, can affect the performance of race dogs. Alternatively, thecomposition comprising a recombinant vector expressing at least oneimmune response-inducing epitope can be applied directly to the skin forlocalized expression and induction of an immune response.

Efficacy of the composition, which can induce an immune response, can bedemonstrated by exposing puppies to a virulent strain of canineinfluenza virus. Untreated dogs should develop clinical signscharacteristic of canine influenza viral infection, whereas treated dogsshould not.

The recombinant vectors and the products expressed from them can be usedto produce antibodies, such as polyclonal antibodies (pAb) andmonoclonal antibodies (mAb), in accordance with methods known in the art(Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1988); Harlow and Lane,Using Antibodies: A Laboratory Manual (1998), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1998); Shepherd and Dean,Monoclonal Antibodies: A Practical Approach, Oxford University Press,U.S.A. (2000)); and Harris and Adair, Antibody Therapeutics, CRC Press,Inc., Boca Raton, Fla. (1997)). The antibodies, in particular mAbs, canbe used in binding assays and diagnostic kits/tests to determine thepresence/absence of an antigen of canine influenza virus or whether ornot an immune response to the virus has been stimulated. The antibodiesalso can be used to recover material by immuno-adsorptionchromatography.

Antibodies also can provide passive immunization. For example, partiallypurified immune sera from host animals or from hybridoma cell lines canbe injected into an animal. The antibodies provide a therapeutic effectby binding to and neutralizing an infectious influenza virus.

A composition comprising an anti-idiotypic antibody having an internalimage of an epitope of an above-described protein, such as a proteinconsisting of the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 3, isalso provided.

One of ordinary skill in the art will appreciate that an anti-idiotypicantibody, which bears an internal image of an epitope, such as thosedescribed herein, can be prepared. See, e.g., Herlyn et al., Science232: 100-102 (1986)). Methods of preparing monoclonal and polyclonalanti-idiotypic antibodies, which bear the internal image of thepolypeptide, are described in U.S. Pat. No. 5,053,224, for example.Briefly, polyclonal anti-idiotypic antibodies can be produced byimmunizing animals with monoclonal idiotypic antibodies raised againstthe polypeptide and screened for reactivity with the polypeptide andscreening for antisera, which react with idiotypic antibodies to thepolypeptide. Monoclonal antibodies (mAbs) also can be prepared from suchanimals using standard techniques of immortalizing theantibody-secreting cells of the animal and screening cultures withidiotypic antibodies in competition with the polypeptide. While mAbs arepreferred, polyclonal antibodies (pAbs), which are prepared in a varietyof mammalian systems, also can be used.

Another method for inducing an immune response to CIV in a canine isalso provided. This method comprises administering to the canine aneffective amount of a composition comprising an anti-idiotypic antibodyas described above.

The isolated or purified nucleic acid molecules or vectors comprisingthem can be used to generate DNA for probes/primers, which can be usedto detect the presence or absence of hybridizable DNA or to amplify DNA,such as cDNA.

Labeled proteins or fragments thereof, as well as labeled nucleic acidsor fragments thereof, can be used in assays. Assay methods includefluoroimmunoassays (smith et al., Ann. Clin. Biochem. 18: 253-275(1981)), radioimmunoassays (RIA), enzyme-linked immunosorbent assays(ELISA), and enzyme-multiplied immunoassay technique (EMIT; see EnzymeImmunoassay, Maggio, ed., CRC Press, Inc., Boca Raton, Fla., 1980. pp.141-150; 234-235, and 242-243). Such methods can be used to detect thepresence of the virus and to diagnose the state of infection.

The virus, itself, can be used as a vector. The use of viruses asvectors is within the skill in the art.

EXAMPLE

The following example serves to illustrate the present invention. Theexample is not intended to limit the scope of the invention in any way.The example describes the identification and partial characterization ofa canine influenza virus.

Outbreaks of acute respiratory disease, characterized by cough, fever,rapid respiration, and hemorrhagic nasal discharge, occurred amonggreyhounds within two race track compounds located in eastern andwestern Iowa in Apr. 2005. While a large percentage of affected dogsrecovered, many succumbed to hemorrhagic pneumonia.

Lungs of affected dogs exhibited extensive red to red-blackdiscoloration with moderate to marked palpable firmness and mildfibrinous pleuritis. Lung sections were characterized by severehemorrhagic interstitial to bronchointerstitial pneumonia. Patchyinterstitial change with alveolar septal thickening, coagulums of debrisin alveoli, and associated atelectasis were evident. Focally extensivepyogranulomatous bronchointerstitial pneumonia with dilatation ofairways by degenerate cells and debris was observed. Scatteredvasculitis and vasular thrombi were apparent.

Microbiological testing for conventional viral and bacterial agents didnot reveal any significant pathogens except Streptococcus equi subsp.zooepidemicus, which was present in lung tissues from all animalsexamined. Two of four lung samples tested positive for influenza virususing real-time reverse transcriptase-polymerase chain reaction (RT-PCR;Harmon et al., Development of a PCR-based differential test for H1N1 andH3N2 swine influenza viruses. In: Proceedings of the 42nd Annual Meetingof American Association of Veterinary Laboratory Diagnosticians. SanDiego, Calif. Oct. 1999. p. 44.). Immunohistochemistry using monoclonalantibody (mAb) specific for the NP of influenza virus (Vincent et al.,J. Vet. Diagn. Invest. 9: 191-195 (1997)) was also positive within viralpneumonic lesions of both lungs as was antigen-capturing ELISA(Directgen™ Flu A, Becton/Dickinson, Sparks, Md.) testing on thesamples. Bronchioalveolar lavage samples from the two positive lungstested positive for influenza virus by PCR.

Virus isolation was attempted because the detection of influenza virusin canine lungs was an unexpected observation, since only a singlereport of influenza virus infection in dogs existed (Dubovi et al.,Isolation of equine influenza virus from racing greyhounds with fatalhemorrhagic pneumonia. In: Proceedings of the 47th Annual Meeting ofAmerican Association of Veterinary Laboratory Diagnosticians.Greensboro, N.C. Oct. 2004. p. 158.). A virus that was able toagglutinate rooster red blood cells was isolated in Madin-Darby caninekidney (MDCK) cells from lung and bronchioalveolar lavage fluid of oneof the two animals in which influenza virus was detected byimmunohistochemical (IHC) assay and PCR. The isolate was determined byPCR to be influenza virus of H3 subtype. The virus isolate was subtypedas H3N8 using HA-inhibition and NM-inhibition assays. The virus isolatewas recognized by antisera raised against various H3 equine influenzaviruses, including Miami ((A/Eq/MI/1/63-H3N8) 640-1280),AK((A/Eq/AK/29759/91-H3N8) 320-640), and Kentucky((A/Eq/Kentucky/81-H3N8) 160-320).

Sequencing of HA and NA genes of both isolates revealed 100% and 99.8%identity, respectively, between the two isolates. Phylogenetically, theHA gene of the isolates was genetically close (96-98% nucleotidehomology) to the HA gene of recent H3N8 equine influenza viruses (Mackenet al., The value of a database in surveillance and vaccine selection.In: Options for the Control of Influenza IV. Osterhaus et al., eds.Elsevier Science, Amsterdam. 2001. pp. 103-106.). The NA gene of theisolates also showed 96-98% homology with the NA gene of recent H3N8equine influenza viruses. Since greyhounds in two different race tracks,which are geographically remote in Iowa, simultaneously succumbed to thedisease without the involvement of sick horses indicates that theinfluenza virus isolate is a canine-adapted strain that can perpetuatein and spread among dogs. S. zooepidemicus, which has been implicated inrespiratory disease and septicemia-associated problems in many differentanimal species (Wood et al., J. Clin. Microbiol. 43: 120-126 (2005); andGillespie et al., The General Staphylococcus and Streptococcus. In:Hagan and Bruner's Infectious Diseases of Domestic Animals. 7th ed.Comstock/Cornell University Press. Ithaca, N.Y. 1981. pp. 164-180)),probably contributed to the severity of the disease.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a,” “an,” “the,” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to illuminate better the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Itshould be understood that the illustrated embodiments are exemplaryonly, and should not be taken as limiting the scope of the invention.

What is claimed is:
 1. An isolated or purified Hemagglutinin HA, which(i) has the amino acid sequence of SEQ ID NO: 4 or (ii) is derived froman influenza virus and which has an amino acid sequence that is greaterthan 99% identical to SEQ ID NO: 4, with the proviso that the amino acidsequence is identical to that of SEQ ID NO: 4 at amino acid positions 94and 233 and the isolated or purified HA has a leucine at position 94 anda glutamic acid at position 233, according to the numbering of SEQ IDNO:
 4. 2. A composition comprising the isolated or purified HA of claim1 in an amount sufficient to induce an immune response in an animal anda biologically acceptable carrier.
 3. A method of inducing an immuneresponse to a canine influenza H3 virus in an animal, which methodcomprises administering to the animal the composition of claim 2,whereupon where upon an immune response to canine influenza H3 virus isinduced in the animal.
 4. An A vector comprising the isolated orpurified nucleic acid encoding the HA of claim 1, optionally as part ofa vector.
 5. The isolated or purified nucleic acid The vector of claim4, wherein the nucleic acid encoding the HA comprises the nucleotidesequence of SEQ ID NO:
 3. 6. A composition comprising the isolated orpurified nucleic acid vector of claim 4, which expresses HA in an amountsufficient to induce an immune response in an animal, and a biologicallyacceptable carrier.
 7. An isolated or purified HA peptide fragmentcomprising a contiguous nine amino acid fragment of SEQ ID NO: 4, or acontiguous nine amino acid fragment of an amino acid sequence that isgreater than 99% identical to SEQ ID NO: 4, that either includes the Leuat position 94 of SEQ ID NO: 4 or the Glu at position 233 of SEQ ID NO:4, according to the numbering of SEQ ID NO:
 4. 8. A compositioncomprising the isolated or purified HA peptide fragment of claim 7 in anamount sufficient to induce an immune response in an animal and abiologically acceptable carrier.
 9. A method of inducing an immuneresponse to a canine influenza H3 virus in an animal, which methodcomprises administering to the animal the composition of claim 8,whereupon where upon an immune response to canine influenza H3 virus isinduced in the animal.
 10. An A vector comprising an isolated orpurified nucleic acid encoding the HA peptide fragment of claim 7,optionally as part of a vector.
 11. A composition comprising theisolated or purified nucleic acid vector of claim 10, which expressesthe HA peptide in an amount sufficient to induce an immune response inan animal, and a biologically acceptable carrier.
 12. An isolatedpolypeptide that is 97% or greater identical to SEQ ID NO: 4 and has aleucine at position 94 and a glutamic acid at position 233, according tothe numbering of SEQ ID NO: 4 and a biologically acceptable carrier. 13.An isolated DNA which encodes a polypeptide that is 97% or greateridentical to SEQ ID NO: 4 and has a leucine at position 94 and aglutamic acid at position 233 according to the numbering of SEQ ID NO:4.
 14. An isolated polypeptide comprising a contiguous nine amino acidsequence that is greater than 97% identical to a sequence fragment ofSEQ ID NO: 4, wherein said sequence fragment comprises either the Leu atposition 94 or the Glu at position 233 of SEQ ID NO: 4, according to thenumbering of SEQ ID NO:
 4. 15. A method of inducing an immune responseto a canine influenza H3 virus in an animal, which method comprisesadministering to the animal the composition of claim 4 or claim 12,where upon an immune response to a canine influenza H3 virus is inducedin the animal.